This invention relates to platelet-derived growth factor (PDGF) analogues and their use as cell antiproliferative agents.
Relevant background material is incorporated herein by reference in the text to the listed references in the appended bibliography.
Platelet-derived growth factor (PDGF) is a potent mitogen for connective tissue cells and promotes the proliferation of fibroblasts and smooth muscle cells (SMC) [333]. The growth factor is a 28-31 KD dimeric, highly basic (Pi=9.8-10) glycoprotein consisting of two highly homologous (up to 60% sequence homology) polypeptide chains which are the products of distinct genes. The gene products designated A (on chromosome 7) and B (on chromosome 22) are assembled to form either a disulphide-linked heterodimer (PDGF-AB) or a homodimer (PDGF-AA or PDGF-BB). Analysis of the PDGF present in human platelets reveals that it is a mixture of all three dimeric forms with AB being the predominant form (up to 70%) [10;12]. The human prot-oncogene, c-sis, which codes for the PDGF-B chain [21] has been identified as the human homologue of the v-sis oncogene of the transforming retrovirus, simian sarcoma virus. This oncogene codes for the protein p28 v-sis which has been identified as PDGF-BB [5].
The cloning and amino acid sequencing of the A and B chains of human PDGF have shown that both chains are synthesised as precursor molecules with hydrophobic leader sequences and both chains undergo proteolytic cleavage at the N-termini during maturation. The B chain is also processed at the C-terminal end [21;20].
The three isoforms of PDGF exert their biological effects by binding with different affinities to two different receptor types, denoted xcex1 and xcex2. Ligand binding induces dimerization of receptors; the A-subunit of PDGF binds to a-receptors whereas the B-subunit binds to both xcex1- and xcex2-receptors [2].
When PDGF diner is treated with reducing agents, the protein loses its biological activity irreversibly, suggesting that the protein conformation is disturbed by reduction of critical disulphide bonds [16]. PDGF has 8 cysteine residues which are highly conserved between the two chains. Six residues are involved in 3 intra-molecular disulphide bonds: Cys-16xe2x80x94Cys-60, Cys-49xe2x80x94Cys-97 and Cys-53xe2x80x94Cys-99. The other two cysteine residues are involved in asymmetrical inter-molecular disulphide bonds, Cys-43xe2x80x94Cys-52 [11].
A systematic analysis of the abilities of different peptides, derived from the PDGF-B chain sequence, to compete with 125I-PDGF-BB for binding to PDGF xcex2-receptors, has led to the identification of two regions in the B-chain corresponding to amino acid residues 35-40 and 78-83 that seem important for receptor binding. A peptide corresponding to the two sequences (ANFLVWxe2x80x94EIVRKKP) (SEQ ID NOS: 12 and 13, respectively) has been found to be effective as an antagonist for PDGF, although detailed analysis has shown the pure peptide to be less active [6].
Site-directed mutagenesis studies, using deletion and substitution mutants of PDGF-BB or of the homologous v-sis gene as well as PDGF-A/B chimeras, have also identified a number of amino acid residues which are important for the biological activity of PDGF. The region Ile-25xe2x80x94Phe-38 has been identified as a binding domain by site directed mutagenesis of the v-sis gene [9]. Amino acid residue Asn-34 has been found to be essential for the PDGF-B-like transforming efficiency of PDGF-A/B chimera [27]. Using a different functional assay, which selects for mutants with reduced binding to both receptor types, Ile-30 and, to a lesser extent, Arg-27 have been shown to be important [3]. Basic polypeptides such as polypeptides such as polylysin and protamine sulphate inhibit PDGF binding to its receptor, suggesting a role for ligand positive charge in the binding interaction. A receptor binding domain has been assigned to a region at the C-terminal end which is rich with basic amino acid, residues Lys-80xe2x80x94Cys-97 [39]. This region contains the sequence Val-78xe2x80x94Arg-79xe2x80x94Lys-80xe2x80x94Lys-81xe2x80x94Pro-82, (SEQ ID NO:14) which is conserved in both the A and B chains, and therefore may be involved in the binding of both chains to PDGF xcex1-receptor. A mutant PDGF-A chain in which the cationic sequence Arg-Lys-Lys has been replaced by the sequence Glu-Glu-Glu displays a marked reduction in both binding affinity for PDGF xcex1-receptor and mitogenic activity in fibroblast cells [7]. Initial studies with neutralizing monoclonal antibodies raised to PDGF-BB indicates that the segment between Thr-20 and Cys-43 represents a surface domain of PDGF-BB and contains amino acid residues involved in receptor binding [22].
Recently, the crystal structure of the homodimeric BB isoform of human recombinant PDGF has been determined [26]. The protein polypeptide chain is folded into two highly twisted anti-parallel pairs of xcex2-strands and contains an unusual knotted arrangement of three intramolecular disulphide bonds. Dimerization leads to the clustering of three surface loops at each end of the elongated dimer, which most probably form the receptor recognition sites. The three loops are: loop I: Ile-25xe2x80x94Leu -38, loop II: Cys-53xe2x80x94Val-58 and loop III: Val-78xe2x80x94Lys-81.
Antibodies to PDGF would be extremely useful in the study of PDGF processing and biosynthesis. It has been difficult to make high avidity antibodies against PDGF, maybe because the molecule is conserved between species and only recently have monoclonal antibodies against PDGF, become available [22;34;12;38]. Rabbit and goat antisera to PDGF have been made to the two chains using protein purified from human platelets or recombinant protein or synthetic peptides, some showing chain specificity and neutralizing activity [28;17;13;37;30]. None of the antibodies raised to peptides however have been capable of recognising the native molecule or able to neutralize its biological activities.
PDGF has been implicated in many biological systems. Originally, the close similarity between PDGF and the transforming factor involved in SSV transformation led to the concept that over-production of the factor was involved in the development of human malignancies [14]. Examination of many tumour cell lines shows that the A and B chains are commonly expressed in such cell lines [15;24]. In general, aberrant expression of PDGF or of PDGF receptors is likely to be involved in the stimulation of the growth of certain tumours. In addition, over-activity of PDGF could also be part of the development of certain non-malignant disorders involving an excess of cell proliferation. Examples include atherosclerosis, where PDGF-induced stimulation of smooth muscle cell proliferation could contribute to the thickening of the intima of affected vessels [32], as well as chronic fibrotic processes, where PDGF could be involved in the stimulation of connective tissue cell proliferation. Ferns et al [8] showed that in a rat experimental model of angioplasty, polyclonal antibodies to PDGF administered intravenously inhibited smooth muscle cell accumulation in the intima of injured arteries, while administration of PDGF induced SMC proliferation in the media by 2-3 fold and, more significantly, increased SMC migration from the media to the intima by 20-fold [19].
However, PDGF does have a normal function. PDGF and PDGF receptors are expressed in embryonic tissues and in the placenta [23;18] which suggests a function for PDGF during development. A role for PDGF in neuronal development has also been proven [25] and PDGF and its receptors are present in the peripheral and central nervous systems [40;36]. PDGF is known to stimulate growth as well as chemotaxis of connective tissue cells and also chemotaxis of inflammatory cells, which suggests a role in wound healing [4;35]. Recently, PDGF has been used in a clinical trial to look at its wound healing capability. Locally applied PDGF stimulates the healing of large bed sores [31]. PDGF xcex2-receptors occur on capillary endothelial cells [29] and PDGF has weak angiogenic activity [29] which may suggest that its stimulatory effect is important in wound healing.
The varied roles of PDGF, both beneficial and adverse, make PDGF agonists and antagonists highly desirable. They can be used as a replacement for PDGF in wound healing or as inhibitors of the adverse effects of PDGF. Antibodies with neutralizing activity, whether to the mitogenic effect of PDGF and/or the chemotactic effect can also be useful as inhibitors of PDGF adverse effects.
Accordingly, in one aspect the present invention provides novel PDGF peptide analogues and compositions consisting of or containing them for use as antiproliferative agents, particularly antiatherosclerotic, antiatherogenetic, anti-inflammatory or antifibrotic agents. The invention also provides such novel PDGF peptide analogues and compositions consisting of or containing them for use as PDGF agonists for use in wound healing.
Particular PDGF analogues according to the present invention are identified in Table 1 hereinbelow (SEQ ID NOS:1-7). Preferably, the PDGF peptide analogues of the invention, as prepared and used in other aspects and embodiments of the invention, are greater than about 90% pure, more preferably greater than about 95% pure, even more preferably greater than about 99% pure.
Pharmaceutical compositions in accordance with the present invention preferably comprise one or more of the PDGF analogues of the invention together with a pharmaceutically acceptable diluent and/or carrier. Suitable carriers/diluents are well known in the art and include saline or other sterile aqueous media, optionally including additional components such as buffer salts and preservatives, or sugars, starches, salts or mixtures thereof.
Peptides according to the present invention may be provided for use in any suitable form appropriate to the protocol of administration and/or the needs of a patient.
Apart from the pharmaceutically acceptable compositions referred to above, the peptides may for example be provided, either singly or in combination, in lyophilized or freeze dried solid forms.
Within the scope of the invention are linked peptides comprising a first analogue selected from the group consisting of GP1, (SEQ ID NO:1) GP2, (SEQ ID NO:1) GP3, (SEQ ID NO:2) GP4, (SEQ ID NO:2) GP9 (SEQ ID NO:3) and GP10 (SEQ ID NO:3) (as identified in Table 1 hereinbelow) and a second peptide analogue selected from the group consisting of GP5, (SEQ ID NO:4) GP6, (SEQ ID NO:4) GP7, (SEQ ID NO:5) GP8, (SEQ ID NO:5) GP21a, (SEQ ID NO:6) GP21 (SEQ ID NO:7) and GP22 (SEQ ID NO:7) (as identified in Table 1 hereinbelow),
The invention further provides the novel PDGF peptide analogues for use in assays and kits for assays.
It is to be understood that within the scope of the present invention are peptide analogues as described and identified herein in which one or more amino acids are substituted with other amino acids, or in which there is inserted a spacer, for example a dithiol group or a diamino group or multiples of amino acid residues, e.g. glycine, as shown in Table 2 hereinbelow, peptides GP11, (SEQ ID NO:8) GP12, (SEQ ID NO:8) GP13 (SEQ ID NO:9) and GP14 (SEQ ID NO:9). The spacer may also be a homo- or hetero-bifunctional crosslinker, for example the heterobifunctional crosslinker N-(4-carboxy-cyclohexyl-methyl)-maleimide, as shown in Table 3 hereinbelow, peptides GP20 and GP23, (SEQ ID NO:10 and 11, respectively) providing generally of course that the essential activity of the peptide remains substantially unchanged.
The invention further provides the synthesis and use of cyclic peptides such as those derived from GP4 (SEQ ID NO:2) and GPB 8 (SEQ ID NO:5) as shown in Table 4 below, peptides GP24 (SEQ ID NO:1) and GP25 (SEQ ID NO:6).
The invention further provides the novel PDGF peptide analogues for use in assays and kits for assays, either in the free form or linked to a carrier molecule such as a protein or a solid particle, as well as modified peptides with e.g. biotin or fluorescein isothiocyanate, such as those shown in Table 5 hereinbelow, peptides GP15, GP16, GP19, GP17 and GP18.
In a second aspect, the present invention provides a method of inhibiting or stimulating cell proliferation, particularly smooth muscle cell, 3T3-fibroblast cell, connective tissue cell or inflammatory cell proliferation, by use or administration, particularly to a host, of an effective amount of a PDGF peptide analogue as defined above.
The invention further provides a method of inhibiting or stimulating PDGF-induced DNA synthesis comprising use or administration, such as to a host, of an effective amount of a PDGF peptide analogue as defined above.
In a further aspect, the present invention provides PDGF peptide analogues as defined above for use in inhibiting or stimulating growth and/or chemotaxis of cells such as those identified above.
In yet a further aspect, the present invention provides the above-defined PDGF peptide analogues, particularly the linked peptide analogues of the invention, for use as immunogens for the production of polyclonal and monoclonal antibodies to PDGF, especially for diagnostic, prognostic and therapeutic uses. Such methods of production of polyclonal and monoclonal antibodies are also within the scope of the invention.
In yet another aspect of the present invention, the novel PDGF analogues are provided for and used in methods of inhibiting PDGF-induced DNA synthesis, for example by use of or administration of an effective amount of one or more of the above defined PDGF peptide analogues.
Administration of peptides of the invention in any of the methods described herein may be via any suitable protocol. Preferably, administration to a host, especially a human host, is by intravenous injection or infusion, and may be systemic or topical. Such administration of peptides of the invention is in such an amount as to give the desired effective result of the peptide""s activity at the intended site. Thus, a quantity which constitutes an xe2x80x9ceffectivexe2x80x9d amount may depend upon various parameters, such as body weight of the patient, degree of activity required, intended site of activity, severity of the condition to be treated or prevented, all of which will be well understood and appreciated by persons skilled in the art.
Generally, an amount (or total amount) of peptide will be administered which gives a concentration in plasma of from about 1 to about 100 mg mlxe2x88x921, more preferably from about 1 to about 10 mg mlxe2x88x921.